Cryopreservation of in vitro model of corneal endothelia by vitrification

As an in vitro model of natural corneal endotheliums, confluent monolayers of bovine corneal endothelial cells (BCECs) in culture were used in two unique vitrification solutions (VSs) to examine effect of vitrification processes on viability of cells. Investigations by microscopy showed that the confluent monolayer of BCECs in culture was close to endothelium in situ both in cell shapes and cell junctions. Observations by cryomicroscopy indicated that both the two VSs can vitrify during cooling at 100°C/min and weak devitrification occurred during warming also at 100°C/min. An optimal adding and removing protocol of the VSs was designed by calculations to maintain volume change of cells within -50%-40%. The toxicity test results of VSs measured by cell counting kit-8 (CCK-8) show that toxic injury of VS2 is more severe than VS1. After preservation by vitrification, cell viabilities are 61.3% and 51.65% respectively for VS1 and VS2. Thus, cryoprotective effect of VS1 is better than that of VS2.